The effect of timing of microinjection of DNA constructs on the efficiency of transgenic embryo production and improved efficiency and quality through combining EGFP as a reporter gene with nuclear transfer techniques were examined. From 12-24 h after insemination, constructs of pCXNeo-EGFP were microinjected into a pronucleus of bovine IVM-IVF zygotes. Due to difficulty in visualizing the pronucleus, the incidence of successful injection of linear DNA into pronucleus was higher when injected from 20 to 24 h, as compared with an early period from 12 to 16 h after insemination. However, developmental competence of DNA-injected zygotes and the EGFP-expression rate were not different among the variously-timed microinjections. A majority of the embryos expressing EGFP signal were mosaic. Following nuclear transfer of blastomeres expressing EGFP, 4.5 % of morulae that developed from the NT-eggs had a strong EGFP signal in all live blastomeres. In other embryos, EGFP signal had been lost. When cells derived from the EGFP-positive NT morulae were subcultured, all the cells expressed strong EGFP signal at the second passage and demonstrated neomycin resistance. These results show that transient expression of non-integrated EGFP appears frequently in EGFP-positive bovine embryos and that additional selection of EGFP-positive morulae after nuclear transfer of EGFP-positive blastomeres would facilitate selection of transgenic embryos.Efficient methods to produce porcine zygotes in vitro for the microinjection of DNA constructs were also developed. Replacement of caffeine with adenosine or fertilization promoting peptide, which induced capacitation but prevented acrosome reaction of boar spermatozoa, reduced the incidence of polyspermic penetration of porcine oocytes without any reduction in penetration.